Gynogenetic or androgenetic production of pluripotent cells and cell lines, and use thereof to produce differentiated cells and tissues

ABSTRACT

Methods for obtaining pluripotent (embryonic stem) cells from parthenogenetic embryos, especially primates, are provided. These cells are useful for producing differentiated cells, tissues and organs, especially human and non-human primate cells, tissues and organs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/761,897 filed Feb. 7, 2013, now issued as U.S. Pat. No.9,249,388; which is continuation application of U.S. application Ser.No. 11/842,026 filed Aug. 20, 2007, now abandoned; which is acontinuation application of U.S. application Ser. No. 10/374,512 filedFeb. 27, 2003, now issued as U.S. Pat. No. 7,951,591; which is acontinuation application of U.S. application Ser. No. 09/995,659 filedNov. 29, 2001, now abandoned; which is a continuation-in-partapplication of U.S. application Ser. No. 09/697,297 filed Oct. 27, 2000,now abandoned; which claims the benefit under 35 USC §119(e) to U.S.Application Ser. No. 60/161,987 filed Oct. 28, 1999, now expired. Thedisclosure of each of the prior applications is considered part of andis incorporated by reference in the disclosure of this application.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.97-35203-4905 awarded by the U.S. Department of Agriculture. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides a novel method for producing pluripotentmammalian cell lines and differentiated cells, tissues and organsderived therefrom. Unlike previous reported methods for producingpluripotent cells, the subject pluripotent cells will be produced bygynogenetic or androgenetic means, i.e., methods wherein the pluripotentcells are derived from oocytes containing only DNA of male or femaleorigin and therefore will comprise all female-derived or male-derivedDNA. In a preferred embodiment, the oocytes will comprise male orfemale-derived DNA of primate origin, e.g., human.

2. Background Information

After ovulation, the oocytes of most mammals remain blocked at thesecond metaphase stage of meiosis and will eventually degenerate unlesssperm penetration takes place. Sperm entry activates a whole series ofevents in the oocyte which leads to fertilization and the development ofa new individual. The earliest changes that can be recognized in theoocyte during activation are the completion of meiosis with the omissionof the second polar body and the release of the cortical granules. Thisis followed by the formation of the male and female pronuclei containingtheir respective haploid sets of chromosomes. A period of DNA synthesisoccurs before the two sets of chromosomes condense and come together onthe mitotic spindle of the first cleavage division. Fertilization iscompleted with the restoration of the diploid complement of chromosomesin the nuclei of the two-cell embryo.

However, it is also known that under certain conditions, which may occurspontaneously in vivo, or in vitro, under controlled conditions, thatoocytes containing DNA of all male or female origin may become activatedand result in the production of an embryo. Typically such embryo doesnot develop into an offspring, but rather stops developing fairly earlyin embryogenesis.

The activation of oocytes and development of embryos that comprise DNAof all male or female origin is typically effected as a means ofstudying embryogenesis. For example, the activation of oocytescontaining DNA of all female origin, without any contribution from themale gamete, and the production of an embryo therefrom, is known as“parthenogenesis.” This method has been used by many researchers as ameans for studying embryogenesis in vitro.

Parthenogenesis is a type of gynogenesis. Gynogenesis broadly is definedas the phenomena wherein an oocyte containing all female DNA becomesactivated and produces an embryo. Gynogenesis includes parthenogenesisas well as activation methods wherein the spermatozoa activates theoocyte to complete meiosis, but fails to contribute any genetic materialto the resulting embryo. As in parthenogenesis, the activated oocytedoes not contain DNA of male origin. However, unlike parthenogenesis,however, the male gamete does make a contribution, i.e., it stimulatesoocyte activation.

Androgenesis can be considered to be the opposite of gynogenesis. Thisrefers to the production and activation of an oocyte containing DNA ofentirely male origin, and the development of an embryo therefrom.

In general, embryos that result from oocytes containing DNA of allfemale or male origin only develop to a certain point, and then stopdeveloping. This is hypothesized to occur, e.g., in the case ofparthenogenetic embryos of the instability of the aging unfertilizedoocyte in the maintenance of the meiotic block. In fact,parthenogenetically activated oocytes may give rise to a variety ofaberrations that occur during the completion of meiosis, which mayresult in the production of embryos of different genetic constitutions.

It is known that artificial activation of mammalian oocytes, includingoocytes containing DNA of all male or female origin, can be induced by awide variety of physical and chemical stimuli. Examples of such methodsare listed in the Table below.

TABLE 1 List of Physical and Chemical Stimuli which Can Induce OocyteActivation in Mammals Physical Chemical 1. Mechanical 1. Enzymatic (a)pricking trypsin, pronase, hyaluronidase (b) manipulation of oocytes invitro 2. Osmotic 2. Thermal 3. Ionic (a) cooling (a) divalent cations(b) heating (b) calcium ionophores 3. Electric 4. Anaesthetics (a)general-ether, ethanol, nembutal, chloroform, avertin (b)local-dibucaine, tetracaine, lignocaine, procaine 5. Phenothiazine,tranquillizers thioridazine, trifluoperazine, fluphenazine,chlorpromazine 6. Protein synthesis inhibitors cycloheximide, puromycin7. Phosphorylation inhibitors (e.g., DMAP) 8. Inisitol1,4,5-triphosphate (Ins P3)

Indeed, the activation of parthenogenetic oocytes has been well reportedin the literature. For example, Ware et al., Gamete Research, 22:265-275(1989) teach the ability of bovine oocytes to undergo parthenogeneticactivation using Ca⁺⁺, Mg⁺⁺-H⁺ ionophore (A23187) or electric shock.Also, Yang et al., Soc. Study Reprod., 46:117 (1992) teach activation ofbovine follicular oocytes using cycloheximide and electric pulsetreatment. Graham C. F. in Biol. Rev., 49:399-422 (1979) describes thenexisting methods for activating parthenogenetic mammalian embryos.Further, Matthew H. Kaufman, in Prog. in Anat., Vol. 1:1-34, ed. R. G.Harrison and R. L. Holmes, Cambridge Press, London, UK (1981) reviewsparthenogenesis and methods of activation. The parthenogeneticactivation of rabbit and mouse oocytes is also disclosed by Ozil, JeanPierre, Devel., 109:117-127 (1990); Kubiak, Jacek, Devel. Biol.,136:537-545 (1989); Onodera et al., Gamete Research, 22:277-283 (1989);Siracusa et al., J. Embryol. Exp. Morphol., 43:157-166 (1978); andSzollosi et al., Chromosoma, 100:339-354 (1991). Still further, theactivation of unfertilized sea urchin eggs is disclosed by Steinhardt etal., Nature, 252:41-43 (1974); and Whitaker, M., Nature, 342:636-639(1984). Also, the parthenogenetic activation of human oocytes has beenreported. (See, e.g., De Sutter et al., J. Associated Reprod. Genet.,9(4):328-336 (1992)).

In general, the goal of such artificial oocyte activation methods hasbeen known in biological research, in particular the study of embryonicdevelopment, and the mechanisms which are involved in oocyte activation.

In recent years, a significant goal of many research groups has been theidentification of methods that efficiently and reproducibly give rise topluripotent or embryonic stem cells (ES cells), and ES or pluripotentcell lines. ES cells are extremely desirable because of theirpluripotency which allows them to give rise to any differentiated celltype. Also, ES cells are useful for the production of chimeric animalsand as an in vitro model for differentiation studies, especially thestudy of genes involved in early development.

Methods for deriving embryonic stem (ES) cell lines in vitro from earlypreimplantation mouse embryos are well known. (See, e.g., Evans et al.,Nature, 29:154-156 (1981); Martin, Proc. Natl. Acad. Sci., USA,78:7634-7638 (1981)). ES cells can be passaged in an undifferentiatedstate, provided that a feeder layer of fibroblast cells (Evans et al.,Id.) or a differentiation inhibiting source (Smith et al., Dev. Biol.,121:1-9 (1987)) is present.

ES cells have been previously reported to possess numerous applications.For example, it has been reported that ES cells can be used as an invitro model for differentiation, especially for the study of genes whichare involved in the regulation of early development. Mouse ES cells cangive rise to germline chimeras when introduced into preimplantationmouse embryos, thus demonstrating their pluripotency (Bradley et al.,Nature, 309:255-256 (1984)).

In view of their ability to transfer their genome to the nextgeneration, ES cells have potential utility for germline manipulation oflivestock animals by using ES cells with or without a desired geneticmodification. Moreover, in the case of livestock animals, e.g.,ungulates, nuclei from like preimplantation livestock embryos supportthe development of enucleated oocytes to term (Smith et al., Biol.Reprod., 40:1027-1035 (1989); and Keefer et al., Biol. Reprod.,50:935-939 (1994)). This is in contrast to nuclei from mouse embryoswhich beyond the eight-cell stage after transfer reportedly do notsupport the development of enucleated oocytes (Cheong et al, Biol.Reprod., 48:958 (1993)). Therefore, ES cells from livestock animals arehighly desirable because they may provide a potential source oftotipotent donor nuclei, genetically manipulated or otherwise, fornuclear transfer procedures.

Some research groups have reported the isolation of purportedlypluripotent embryonic cell lines. For example, Notarianni et al., J.Reprod. Fert. Suppl., 43:255-260 (1991), report the establishment ofpurportedly stable, pluripotent cell lines from pig and sheepblastocysts which exhibit some morphological and growth characteristicssimilar to that of cells in primary cultures of inner cell massesisolated immunosurgically from sheep blastocysts. Also, Notarianni etal., J. Reprod. Fert. Suppl., 41:51-56 (1990) disclose maintenance anddifferentiation in culture of putative pluripotential embryonic celllines from pig blastocysts. Gerfen et al., Anim. Biotech, 6(1):1-14(1995) disclose the isolation of embryonic cell lines from porcineblastocysts. These cells are stably maintained in mouse embryonicfibroblast feeder layers without the use of conditioned medium, andreportedly differentiate into several different cell types duringculture.

Further, Saito et al., Roux's Arch. Dev. Biol., 201:134-141 (1992)report cultured, bovine embryonic stem cell-like cell lines whichsurvived three passages, but were lost after the fourth passage.Handyside et al., Roux's Arch. Dev. Biol., 196:185-190 (1987) discloseculturing of immunosurgically isolated inner cell masses of sheepembryos under conditions which allow for the isolation of mouse ES celllines derived from mouse ICMs. Handyside et al. report that under suchconditions, the sheep ICMs attach, spread, and develop areas of both EScell-like and endoderm-like cells, but that after prolonged culture onlyendoderm-like cells are evident.

Cherny et al., Theriogenology, 41:175 (1994) reported purportedlypluripotent bovine primordial germ cell-derived cell lines maintained inlong-term culture. These cells, after approximately seven days inculture, produced ES-like colonies which stained positive for alkalinephosphatase (AP), exhibited the ability to form embryoid bodies, andspontaneously differentiated into at least two different cell types.These cells also reportedly expressed mRNA for the transcription factorsOCT4, OCT6 and HES1, a pattern of homeobox genes which is believed to beexpressed by ES cells exclusively.

Campbell et al., Nature, 380:64-68 (1996) reported the production oflive lambs following nuclear transfer of cultured embryonic disc (ED)cells from day nine ovine embryos cultured under conditions whichpromote the isolation of ES cell lines in the mouse. The authorsconcluded that ED cells from day nine ovine embryos are totipotent bynuclear transfer and that totipotency is maintained in culture.

Van Stekelenburg-Hamers et al., Mol. Reprod. Dev., 40:444-454 (1995),reported the isolation and characterization of purportedly permanentcell lines from inner cell mass cells of bovine blastocysts. The authorsisolated and cultured ICMs from 8 or 9 day bovine blastocysts underdifferent conditions to determine which feeder cells and culture mediaare most efficient in supporting the attachment and outgrowth of bovineICM cells. They concluded that the attachment and outgrowth of culturedICM cells is enhanced by the use of STO (mouse fibroblast) feeder cells(instead of bovine uterus epithelial cells) and by the use ofcharcoal-stripped serum (rather than normal serum) to supplement theculture medium. Van Stekelenburg et al. reported, however, that theircell lines resembled epithelial cells more than pluripotent ICM cells.

Smith et al., WO 94/24274, published Oct. 27, 1994, Evans et al., WO90/03432, published Apr. 5, 1990, and Wheeler et al., WO 94/26889,published Nov. 24, 1994, report the isolation, selection and propagationof animal stem cells which purportedly may be used to obtain transgenicanimals. Evans et al. also reported the derivation of purportedlypluripotent embryonic stem cells from porcine and bovine species whichassertedly are useful for the production of transgenic animals. Further,Wheeler et al., WO 94/26884, published Nov. 24, 1994, disclosedpurported embryonic stem cells which are assertedly useful for themanufacture of chimeric and transgenic ungulates. However, to the bestof the inventors' knowledge, this work has never been reported in anypeer-reviewed journal.

Quite recently, two research groups simultaneously reported theproduction of purified hon-human primate and human ES cells. These EScell lines were derived from non-human primate and human embryos. Thesepurported ES cell lines were reported to be SSEA-1 negative, SSEA-4, andSEA-3 positive, TRA-1-60 and TR-A-1-81 positive, and alkalinephosphatase positive, to develop into all three embryonic germ layers(endoderm, mesoderm, ectoderm), to maintain a normal karyotype evenafter prolonged culturing, and to proliferate indefinitely in vitro inan undifferentiated state.

Also recently, a group of scientists at the University of Massachusettsand Advanced Cell Technology reported the production of a “human embryo”by cross species nuclear transplantation of human differentiated cellsinto a bovine oocyte, and the potential use thereof for the productionof human ES cells. However, notwithstanding what has been reported,improved methods for obtaining ES cells, and methods for maintainingsuch cells in vitro for indefinite periods would be extremely useful.

OBJECTS OF THE INVENTION

Therefore, it is an object of the invention to provide a novel methodfor producing pluripotent cells and cell lines, i.e., ES cells.

More specifically, it is an object of the invention to provide a methodfor obtaining pluripotent cells or cell lines that comprisefemale-derived or male-derived DNA.

Still more specifically, it is an object of the invention to provide amethod for obtaining pluripotent cells or cell lines that compriseprimate female-derived or male-derived DNA, male or female DNA of humanorigin.

Related thereto, it is an object of the invention to provide pluripotentcells or cell lines that are isogenic to male or female donors, e.g., ahuman donor.

Also, it is an object of the invention to utilize pluripotent cells thatcomprise all female-derived or male-derived DNA for the production ofdifferentiated cells, tissues and organs.

It is a related object of the invention to use pluripotent cells or celllines, preferably human pluripotent cells or cell lines, to produceprimate, preferably human, differentiated cells, tissues or organs byinducing differentiation of pluripotent cells or cell lines thatcomprise DNA of all male or female origin.

Further, it is an object of the invention to utilize pluripotent cellsthat comprise all female-derived or male-derived DNA for the productionof chimeric animals.

It is an even more specific object of the invention to producepluripotent cells containing all male-derived or female-derived DNA bythe following method:

-   -   (i) producing a cell preferably in metaphase II, preferably an        oocyte or blastomere that comprises all male- or female-derived        DNA;    -   (ii) activating such oocyte or blastomere under conditions        that (1) does not result in second polar body extrusion or (2)        inhibits polar body extrusion, or (3) inhibits the first        cleavage event, thereby producing an activated oocyte having a        diploid content of DNA of male or female origin;    -   (iii) culturing the resultant activated oocyte or blastomere        under conditions that give rise to an embryo comprising a        trophectoderm and inner cell mass; and    -   (iv) culturing cells derived from said inner cell mass in vitro        under conditions that maintain said cells in an        undifferentiated, pluripotent state.

It is another more specific object of the invention to producepluripotent cells containing all male-derived or female-derived DNA bythe following method:

-   -   (i) transplanting an enucleated oocyte or blastomere with two        haploid nuclei, both of either male or female origin;    -   (ii) activating said oocyte or blastomere;    -   (iii) culturing said activated oocyte or blastomere under        conditions that result in an embryo comprising a trophectoderm        and inner cell mass; and    -   (iv) culturing cells obtained from said inner cell mass in vitro        under conditions that maintain said cells in an        undifferentiated, pluripotent state.

It is another object of the invention to (i) produce embryos containingDNA of all male or female origin by any of the above-identified methodswhich contains a discernible inner cell mass and trophectoderm, (ii)selecting cells from said inner cell mass or cells which have beenisolated from said inner cell mass and cultured under conditions thatmaintain said cells in an undifferentiated, pluripotent state; and (iii)to confirm the pluripotency of such selected cells.

This can be effected by various methods, e.g., by testing for thepresence of certain molecular markers that are characteristic ofpluripotency; by making chimeric animals using said cells and testingfor the genetic contribution of said cells to the different cells of thechimeric animal by injection into SCID mice and observing the productionof different differentiated cell types, and by observing thedifferentiation of such cells into embryoid bodies and other types ofdifferentiated cells in tissue culture.

It is another object of the invention to provide a theoreticallyinfinite supply of isogenic pluripotent cells by: (i) producing anembryo derived from all male or female DNA that contains a discernibletrophectoderm and inner cell mass; (ii) culturing at least the innercell mass of said embryo or a portion thereof under conditions thatmaintain said cells in a pluripotent undifferentiated state. This ispreferably effected by culturing the inner cell mass or a portionthereof under conditions whereby such cells or inner cell mass ismaintained in contact with a feeder layer, e.g., fetal fibroblasts.These cultured pluripotent cells will be transferred to new feederlayers as often as is required to maintain said cells in the desiredpluripotent state.

Another object of the invention is to produce pluripotent cells thatcomprise a desired DNA modification. This will be effected by producingoocytes containing DNA of all male or female origin, wherein said DNAcomprises a desired modification; activating said oocytes underconditions that give rise to an embryo having a discernibletrophectoderm and inner cell mass; and culturing cells from said innercell mass or the entire cell mass under conditions that inhibitdifferentiation and maintain said cells in a pluripotent state thatcontain the desired DNA modification.

Still another object of the invention is to produce differentiated cellsor tissues that contain a desired DNA modification comprising:

-   -   (i) producing oocytes that contain DNA of all male or female        origin, which DNA comprises at least one modification;    -   (ii) activating said oocytes under conditions that give rise to        an embryo containing a discernible trophectoderm and inner cell        mass;    -   (iii) culturing at least said inner cell mass or a portion        thereof under conditions that maintain said cells in an        undifferentiated, pluripotent state; and    -   (iv) using said cultured cells to produce differentiated cells        containing said at least one DNA modification.        This can be effected, e.g., by altering the cell culture        procedure, e.g., by addition of growth factors that promote        differentiation; by removal of feeder layers, by injection into        a suitable animal, e.g., SCID mouse, whereby differentiated        cells and/or tissues arise from said cells in vivo; or by the        use thereof to produce a chimeric animal or fetus that contains        differentiated cells that express the genotype of said male or        female DNA, which comprises at least one DNA modification.

Still another object of the invention is to provide cell culturescomprising pluripotent cells which optionally may be geneticallymodified, wherein said pluripotent cells are derived from embryosproduced by androgenetic or gynogenetic methods.

Yet another object of the invention is to provide differentiated cellsand tissues derived from pluripotent cells which are themselves derivedfrom embryos produced by androgenetic or gynogenetic methods.

Still another object of the invention is to use said pluripotent cells,or differentiated cells derived therefrom, which optionally may begenetically modified for cell and gene therapy, tissue and organtransplantation, or for the study of embryogenesis and differentiation.For example, the effect of specific DNA modifications on embryonicdevelopment or the production of specific types of differentiated cellscan be effected.

Yet another object of the invention is to culture pluripotent cells andcell lines produced according to the invention with differentcombinations of hormones, cytokines, and growth factors and ratiosthereof that induce the differentiation of the subject pluripotent cellsinto particular differentiated cell types.

It is yet another object of the invention to provide cloned animals,having a particular genotype, by use of pluripotent cells, ordifferentiated cells provided therefrom, produced according to theinvention as nuclear donors for nuclear transfer.

It is still another object of the invention to utilize sperm or oocytesfrom animals having desired characteristics, e.g., preferredagricultural characteristics, for the production of pluripotent cellsand cell lines. These pluripotent cells or differentiated cells may beutilized as nuclear transfer donors.

It is another object of the invention to provide a novel business methodfor producing cloned agricultural animals having a desired genotype bysperm or oocytes derived therefrom for use in the production ofpluripotent cells or cell lines having the same genotype.

In a preferred object of the invention, the pluripotent cells ordifferentiated cells derived therefrom will be obtained by activation ofan oocyte that contains only DNA of human male or human female origin,which DNA is optionally genetically modified to produce an embryocontaining a discernible trophectoderm and inner cells mass, and whereinthe inner cell mass or a portion thereof is cultured under conditionsthat maintain said cells in a pluripotent undifferentiated state; andwhich pluripotent cells give rise to differentiated cell types in vivo(e.g., in a SCID mouse), or when cultured under appropriate conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Embryoid body photographed at 100× magnification. Formed fromexplant plated directly on culture dish (no mouse fetal fibroblastfeeder layer). Original colony (1023981-3) plated from a blastocystactivated previously. Light is deflected showing lipid content of cells.

FIG. 2: Embryoid body photographed at 100× magnification. Formed fromexplant plated directly on culture dish (no mouse fetal fibroblastfeeder layer). Original colony (1023981-3) plated from a blastocystactivated a week prior.

FIG. 3: Embryoid body photographed at 100× magnification. Formed fromexplant plated directly on culture dish (no mouse fetal fibroblastfeeder layer). Original colony (1023981-3) plated from a blastocystactivated week prior.

FIG. 4: Edge of explanted stem cell colony photographed at 40×magnification. Original colony (1023981-3) plated from blastocystactivated week prior. Stem cell colony is top left and mouse fetalfibroblast feeder layer is bottom right.

FIG. 5: Edge of explanted stem cell colony photographed at 100×magnification. Original colony (1023981-3) plated from blastocystactivated week prior. Stem cell colony is top left and mouse fetalfibroblast feeder layer is bottom right.

FIG. 6: Center of explanted stem cell colony photographed at 100×magnification. Original colony plated (1023981-3) from blastocystactivated week prior.

FIG. 7: Center of explanted stem cell colony photographed at 200×magnification. Original colony plated (1023981-3) from blastocystactivated week prior.

FIG. 8: Edge of explanted stem cell colony photographed at 40×magnification. Original colony (0106992-2) plated from blastocystactivated week prior. Stem cell colony is top right. Mouse fetalfibroblast feeder layer is bottom left. Light is deflected showingdifference in lipid content between the cells of the stem cell colonyand the mouse fibroblast feeder layer.

FIG. 9: Edge of explanted stem cell colony photographed at 40×magnification. Original colony (0106992-2) plated from blastocystactivated week prior. Stem cell colony is on the top. Mouse fetalfibroblast feeder layer is on the bottom. Light is deflected showingdifference in lipid content between the cells of the stem cell colonyand the mouse fibroblast feeder layer.

FIG. 10: Edge of explanted stem cell colony photographed at 200×magnification. Original colony (0106992-2) plated from blastocystactivated week prior. Stem cell colony is on the left. Mouse fetalfibroblast feeder layer is on the right. Photograph showsdifferentiation of the cells at the edge of the stem cell colony.

FIG. 11 shows three monkey blastocysts on day 8 of development.

FIG. 12 shows a cell line (Cyno 1) obtained from one of the three monkeyblastocysts shown in FIG. 11, before immunosurgery.

FIG. 13 shows the Cyno 1 cell line after immunosurgery.

FIG. 14 shows the Cyno 1 cell line 5 days after plating on a fibroblastfeeder layer.

FIG. 15 shows a pluripotent cell line (referred to as Cyno 1) derivedfrom a parthogenetically activated Cynomolgous monkey oocyte, growing ona mouse fibroblast layer. The feeder cells exhibit morphologicalcharacteristics of pluripotent cells, e.g., small nuclear cytoplasmicratios and detectable cytoplasmic granules.

FIG. 16 shows RT-PCR results demonstrating differentiation of multiplesomatic cell types in differentiating PPSCs: Brachy: Brachyury (T)protein, MYH2: Skeletal myosin Heavy Polypeptide 2, Enolase: Humanneuron-specific Enolase 2, SHH: Human homolog of Sonic Hedgehog.

FIG. 17 shows RT-PCR results using mRNA from monkey fibroblasts with orwithout reverse transcriptase and PPSCs with or without reversetranscriptase. A PCR product was detected of the predicted length onlyin the monkey fibroblasts cells.

DETAILED DESCRIPTION OF THE INVENTION

Prior to describing the invention in detail, the following terms aredefined. Otherwise, all terms in this application have their ordinaryart-recognized meaning.

A. Definitions

Gynogenesis—in the present invention this refers to the production of anembryo containing a discernible trophectoderm and inner cell mass thatresults upon activation of a cell, preferably an oocyte, or otherembryonic cell type, containing mammalian DNA of all female origin,preferably human female origin, e.g., human or non-human primate oocyteDNA. Such female mammalian DNA may be genetically modified, e.g., byinsertion, deletion or substitution of at least one DNA sequence, or maybe unmodified. For example, the DNA may be modified by the insertion ordeletion of desired coding sequences, or sequences that promote orinhibit embryogenesis. Typically, such embryo will be obtained by invitro activation of an oocyte that contains DNA of all female origin.Gynogenesis is inclusive of parthenogenesis which is defined below. Italso includes activation methods wherein the sperm or a factor derivedtherefrom initiates or participates in activation, but the spermatozoalDNA does not contribute to the DNA in the activated oocyte.

Androgenesis—in the present invention this refers to the production ofan embryo containing a discernible trophectoderm and inner cell massthat results upon activation of an oocyte or other embryonic cell type,e.g., blastomere, that contains DNA of all male origin, e.g., humanspermatozoal DNA. Optionally, said DNA of all male origin may begenetically modified, e.g., by the addition, deletion, or substitutionof at least one DNA sequence (as described above with respect togynogenesis).

Parthenogenesis—the process by which activation of the oocyte occurs inthe absence of sperm penetration. In the present invention,parthenogenesis refers to the development of an early stage embryocomprising trophectoderm and inner cell mass that is obtained byactivation of an oocyte or embryonic cell, e.g., blastomere, comprisingDNA of all female origin.

Pluripotent cell—in the present invention this refers to a cell derivedfrom an embryo produced by activation of a cell containing DNA of allfemale or male origin that can be maintained in vitro for prolonged,theoretically indefinite period of time in an undifferentiated state,that can give rise to different differentiated tissue types, i.e.,ectoderm, mesoderm, and endoderm. The pluripotent state of said cells ispreferably maintained by culturing inner cell mass or cells derived fromthe inner cell mass of an embryo produced by androgenetic or gynogeneticmethods under appropriate conditions, preferably by culturing on afibroblast feeder layer or another feeder layer or culture that includesleukemia inhibitory factor. The pluripotent state of such cultured cellscan be confirmed by various methods, e.g., (i) confirming the expressionof markers characteristic of pluripotent cells; (ii) production ofchimeric animals that contain cells that express the genotype of saidpluripotent cells; (iii) injection of cells into animals, e.g., SCIDmice, with the production of different differentiated cell types invivo; and (iv) observation of the differentiation of said cells (e.g.,when cultured in the absence of feeder layer or LIF) into embryoidbodies and other differentiated cell types in vitro.

Diploid cell—in the present invention, this typically refers to a cell,e.g., an oocyte or blastomere, having a diploid DNA content of all maleor female origin.

Haploid cell—in the present invention, this typically refers to a cell,e.g., an oocyte or blastomere having a haploid DNA content, wherein thehaploid DNA is of all male or female origin.

Activation—process wherein fertilized or unfertilized oocyte, preferablyin metaphase II of meiosis undergoes a process typically includingseparation of the chromatid pairs, extrusion of the second polar body,resulting in an oocyte having a haploid number of chromosomes, each withone chromatid. In the present invention, activation refers to methodswhereby a cell containing DNA of all male or female origin is induced todevelop into an embryo that has a discernible inner cell mass andtrophectoderm, which is useful for producing pluripotent cells but whichis itself incapable of developing into a viable offspring. Preferably,in the present invention, activation is preferably effected under one ofthe following conditions: (i) conditions that do not cause second polarbody extrusion; (ii) conditions that cause polar body extrusion butwherein polar body extrusion is inhibited; or (iii) conditions thatinhibit first cell division of haploid oocyte. Also, the presentinvention includes activation of oocytes or blastomeres that have beentransplanted with two male or two female haploid nuclei.

Metaphase II—stage of cell development wherein the DNA content of a cellconsists of a haploid number of chromosomes with each chromosomerepresented by two chromatids.

Embryo—in the present invention this typically refers to an embryo thatresults upon activation of a cell, e.g., oocyte or other embryonic cellscontaining DNA of all male or female origin, which optionally may bemodified, that comprises a discernible trophectoderm and inner cellmass, which cannot give rise to a viable offspring and wherein the DNAis of all male or female origin. The inner cell mass or cells containedtherein are useful for the production of pluripotent cells as definedpreviously.

Inner cell mass—inner portion of an embryo which gives rise to fetaltissues. Herein, these cells are used to provide a continuous source ofpluripotent cells in vitro. In the present invention, the inner cellmass refers to the inner portion of the embryo that results fromandrogenesis or gynogenesis, i.e., embryos that result upon activationof cells containing DNA of all male or female origin. Preferably, suchDNA will be human DNA, e.g., human oocyte or spermatozoal DNA, whichoptionally has been genetically modified.

Trophectoderm—other portion of early stage embryo which gives rise toplacental tissues. In the present invention, the trophectoderm is thatof an embryo that results from androgenesis or gynogenesis, i.e.,embryos that result from activation of cells that contain DNA of allmale or female origin, e.g., human oocyte or spermatozoan.

Differentiated cell—a non-embryonic cell that possesses a particulardifferentiated, i.e., non-embryonic state. The three earliestdifferentiated cell types are endoderm, mesoderm and ectoderm.

B. Detailed Description

The present invention provides novel methods for obtaining pluripotentcells and cell lines, wherein the DNA is derived from a singleindividual, i.e., male or female, by androgenesis or gynogenesis.

In the native environment, immature oocytes (eggs) from the ovaryundergo a process of maturation which results in the progression throughmeiosis to metaphase II of meiosis. The oocytes then arrest at metaphaseII. In metaphase II, the DNA content of the cell consists of a haploidnumber of chromosomes, each represented by two chromatids.

Normally, the oocyte is ovulated at this stage and fertilized by thesperm. The sperm initiates the completion of meiosis in a process calledactivation. During activation, the pairs of chromatids separate, thesecond polar body is extruded, and the oocyte retains a haploid numberof chromosomes, each with one chromatid. The sperm contributes the otherhaploid complement of chromosomes to make a full diploid cell withsingle chromatids. The chromosomes then progress through DNA synthesisduring the first cell cycle. These cells then develop into embryos.

By contrast, in the present invention, embryos are developed byartificial activation of cells, typically mammalian oocytes orblastomeres containing DNA of all male or female origin. As discussed inthe background of the invention, many methods have been reported in theliterature for artificial activation of unfertilized oocytes. Suchmethods include physical methods, e.g., mechanical methods such aspricking, manipulation or oocytes in culture, thermal methods such ascooling and heating, repeated electric pulses, enzymatic treatments,such as trypsin, pronase, hyaluronidase, osmotic treatments, ionictreatments such as with divalent cations and calcium ionophores, the useof anaesthetics such as ether, ethanol, tetracaine, lignocaine,procaine, phenothiazine, tranquilizers such as thioridazine,trifluoperazine, fluphenazine, chlorpromazine, the use of proteinsynthesis inhibitors such as cycloheximide, puromycin, the use ofphosphorylation inhibitors, e.g., protein kinase inhibitors such asDMAP, combinations thereof, as well as other methods.

Such activation methods are well known in the art and are discussed inour earlier patent application U.S. Ser. No. 08/781,752, now U.S. Pat.No. 5,945,577. This application is incorporated by reference in itsentirety herein.

In the present invention, a mammalian cell, preferably in metaphase II,typically an oocyte or blastomere comprising DNA of all male or femaleorigin is artificially activated by a known means for effectingartificial activation of oocytes or nuclear transfer fusions. Dependentupon the activation procedure, the cell may extrude the second polarbody or retain the second polar body.

In one embodiment of the present invention, a mammalian cell, preferablyin metaphase II, e.g., a mammalian oocyte containing haploid content ofDNA of all male or female origin is activated by an activation procedurethat does not result in second polar body extrusion. This can beeffected by various methods including the use of a phosphorylationinhibitor such as DMAP or by use of a microfilament inhibitor such ascytochalasin B, C or D, or a combination thereof. Thereby, cells areobtained having a diploid content of DNA of either male or female originwhich develop into an embryo having a discernible trophectoderm andinner cell mass which will not give rise to viable offspring. The innercell mass or cells therein are used to produce pluripotent cellscontaining cultures which are themselves useful for makingdifferentiated cells and tissues.

In a second embodiment of the invention, a haploid cell, preferably inmetaphase II, typically an oocyte or blastomere that comprises all maleor female DNA, will be activated under conditions that result in theproduction of an embryo having a discernible trophectoderm and innercell mass, but wherein the first cleavage event is prevented, therebyresulting in a diploid cell which develops into an embryo that cannotgive rise to an offspring.

Still alternatively, the invention includes the embodiment wherein anenucleated cell, e.g., mammalian oocyte or blastomere, or othermammalian cytoplast, is transplanted with two male or female haploidnuclei, e.g., derived from oocytes or sperm, and is activated by anappropriate activation procedure to produce an embryo containing adiscernible trophectoderm and inner cell mass which is incapable ofgiving rise to an offspring. Again, the inner cell mass or cells derivedtherefrom are useful for obtaining pluripotent cells which may bemaintained for prolonged periods in tissue culture.

In all cases, the activated cell, e.g., oocyte, which is diploid, isallowed to develop into an embryo that comprises a trophectoderm and aninner cell mass. This can be effected using known methods and culturemedia that facilitate blastocyst development. Examples thereof aredisclosed in our earlier patent application, U.S. Pat. No. 5,945,577,and have been well reported in the literature. Culture media suitablefor culturing and maturation of embryos are well known and include Ham'sF-10+10% fetal calf serum, Tissue Culture Medium, 199 (TCM-199)+10%fetal calf serum, Tyrodes—Albumin—Lactate—Pyruvate (TALP), Dulbecco'sPhosphate Buffered Saline (PBS), Eaglets and Whitten's media, and CR1medium. A preferred medium is for bovine embryos TCM-199 with Earlsalts, 10% fetal calf serum, 0.2 mM Na pyruvate and 50 μg/ml gentamycinsulfate. A preferred medium for culturing pig embryos is NCSU23.

Preferred medium for culturing primate embryos, e.g., human and nonhumanprimate embryos, include modified Ham's F-10 medium (Gibco, Catalog No.430-1200 EB) supplemented with 1 ml/L synthetic serum replacement(SSR-2, Medl-Cult Denmark), and 10 mg/ml HSA; 80% Dulbecco's modifiedEaglet's medium (DMEA, no pyruvate, high glucose formulation, Gibco BRL)with 20% fetal bovine serum, 0.1 mM B-mercaptoethanol, and 1%non-essential amino acid stock, and by methods and medium disclosed inJones et al., Human Reprod. 13(1):169-177 (1998); Thomson et al., Proc.Natl. Acad. Sci., USA, 92:7894-7898 (1995); and Thomson et al., Science,282:1145-1147 (1998); and two media available from Irvine Scientific,Santa Anna, Calif., i.e., a first media called P-1 (Cat. #99242), whichis used for the first three days of culture followed by a second media,P-2 (Cat. #99292) until blastocyst stage.

Suitable activation conditions for human oocytes which may be applicableherein include the following:

1. Activation by Ionomycin and DMAP

(i)

-   -   a. Place oocytes in Ionomycin (5 μM) with 2 mM of DMAP for four        minutes.    -   b. Move the oocytes into culture media with 2 mM of DMAP for        four hours,    -   c. Rinse four times and place in culture.

(ii)

-   -   a. Place oocytes 22 to 28 hours post-maturation in 2 mM of DMAP.    -   b. After one hour, transfer to 5 μM of ionomycin.    -   c. After 3 to 5 minutes, transfer and incubate in 5 μg/ml of        cytochalasm B and 10 μg/ml of cyloheximide for eight hours.

2. Activation by Ionomycin DMAP and Roscovitin

-   -   a. Place oocytes in Ionomycin (5 μm) with 2 mM of DMAP for four        minutes.    -   b. Move the oocytes into culture media with 2 mM of DMAP and 200        microM of Roscovitin for three hours.    -   c. Rinse four times and place in culture.

3. Activation by Exposure to Ionomycin followed by Cytochalasin andCycloheximide

-   -   a. Place oocytes in Ionomycin (5 microM) for four minutes.    -   b. Move oocytes to culture media containing 5 μg/ml of        cytochalasin B and 5 μg/ml of cycloheximide for five hours.    -   c. Rinse four times and place in culture.

4. Activation by Electrical Pulses

-   -   a. Place eggs in mannitol media containing 100 μM CaCL₂    -   b. Deliver three pulses of 1.0 kVcm⁻¹ for 20 μsecond, each pulse        22 minutes apart.    -   c. Move oocytes to culture media containing 5 μg/ml of        cytochalasin B for three hours.

5. Activation by Exposure with Ethanol followed by Cytochalasin andCycloheximide

-   -   a. Place oocytes in 7% ethanol for one minute.    -   b. Move oocytes to culture media containing 5 μg/ml of        cytochalasin B and 5 μg/ml of cycloheximide for five hours.    -   c. Rinse four times and place in culture.

6. Activation by Microinjection of Adenophostin

-   -   a. Inject oocytes with 10 to 20 picoliters of a solution        containing 10 μM of adenophostin.    -   b. Place oocytes in culture.

7. Activation by Microinjection of Sperm Factor

-   -   a. Inject oocytes with 10 to 20 picoliters of sperm factor        isolated either from primates, pigs, bovine, sheep, goat, horse,        mice, rat, rabbit or hamster.    -   b. Place eggs in culture.

8. Activation by Microinjection of Recombinant Sperm Factor

After the androgenetic or gynogenetic embryos have been cultured toproduce a discernable trophectoderm and inner cell mass, the cells ofthe inner cell mass are then used to produce the desired pluripotentcell lines. This can be accomplished by transferring cells derived fromthe inner cell mass or the entire inner cell mass onto a culture thatinhibits differentiation. This is preferably effected by transferringsaid inner cell mass cells onto a feeder layer that inhibitsdifferentiation, e.g., fibroblasts or epithelial cells, such asfibroblasts derived from murines, ungulates, chickens, such as mouse orrat fibroblasts, 570 and SI-m220 feeder cells, BRL cells, etc., or othercells that produce LIF. Preferably, the inner cell mass cells arecultured on mouse fetal fibroblast cells or other cells which produceleukemia inhibitory factor, or in the presence of leukemia inhibitoryfactor. Culturing will be effected under conditions that maintain saidcells in an undifferentiated, pluripotent state, for prolonged periods,theoretically indefinitely.

Suitable conditions for culturing pluripotent cells, specificallypluripotent cells derived from ungulate inner cell mass are alsodescribed in our earlier patent U.S. Pat. No. 5,945,577, as well as U.S.Pat. No. 5,905,042, both of which are incorporated by reference hereinin their entirety.

As noted, the subject invention will give rise to pluripotent cellshaving DNA that is exclusively of male or female origin, which may beused to produce different differentiated cell types.

In a preferred embodiment, such exclusively male or female DNA will begenetically modified before or after activation of the cell containingsame, e.g., human oocyte.

Methods and materials for effecting genetic modification are well knownand include microinjection, the use of viral DNAs, homologousrecombination, etc. Thereby, pluripotent cells are obtained thatcomprise a desired DNA modification, e.g., contain a desired codingsequence.

The novelty of the invention is in the production of pluripotent cellsthat contain either female- or male-derived DNA. To the inventors'knowledge, this has not been reported for any species. Considerable workhas been done in the mouse with pluripotent and totipotent embryonicstem cells but these have all been derived from either normal embryoniccells or primordial germ cells. Each of these cell types has bothpaternal and maternal DNA. Work has also been done with embryoscontaining either male-derived or female-derived DNA. These studiesindicate that for full development of an embryo to term the cells mustcontain both male- and female-derived DNA. Imprinting of the DNA duringmeiosis results in some chromosomal segments being inactivated.Interestingly, if the cells are combined with normal embryonic cells ina chimera they do have the ability to form all the various cell types inthe body. What has not been done for any species is the derivation ofpluripotent cell lines containing only female- or male-derived DNA.Furthermore, no one has shown that these cells are capable ofdifferentiation in culture.

An important feature of these cells is they would be immunologicallysimilar, although not identical to the individual from which the DNAcame. Consequently, they would also be the most immunologicallycompatible cells that could be obtained from an individual without theuse of cloning. Furthermore, they would not generate the ethicalconcerns that the use of a normal embryo would generate because theycould contain DNA from a single individual and would not be capable offorming an offspring.

However, another contemplated application of these cells and cell linesand differentiated cells derived therefrom is as donor cells for nucleartransfer, e.g., by the methods reported in U.S. Pat. No. 5,945,577 orpatents and patent applications by the Roslin Institute. Essentially,these methods differ in that the ACT cloning methods utilizeproliferating donor cells, e.g., mammalian somatic cells, such asfibroblasts, whereas the Roslin Institute methods utilize quiescentdonor cells.

The pluripotent state of the cells produced by the present invention canbe confirmed by various methods.

For example, the cells can be tested for the presence or absence ofcharacteristic ES cell markers. In the case of human ES cells, examplesof such markers are identified supra, and include SSEA-4, SSEA-3,TRA-1-60 and TRA-1-81 and are known in the art.

Also, pluripotency can be confirmed by injecting the cells into asuitable animal, e.g., a SCID mouse, and observing the production ofdifferentiated cells and tissues. Still another method of confirmingpluripotency is using the subject pluripotent cells to generate chimericanimals and observing the contribution of the introduced cells todifferent cell types. Methods for producing chimeric animals are wellknown in the art and are described in our related applications,incorporated by reference herein.

Yet another method of culturing pluripotency is to observe theirdifferentiation into embryoid bodies and other differentiated cell typeswhen cultured under conditions that favor differentiation (e.g., removalof fibroblast feeder layers). This method has been utilized in thepresent invention and it has been confirmed that the subject pluripotentcells give rise to embryoid bodies and different differentiated celltypes in tissue culture. For example, it has been shown that Cynomolgouspluripotent cell lines produced herein give rise to beatingcardiomyoctes and other cells when allowed to differentiate by culturingof the cell line beyond confluency.

The resultant pluripotent cells and cell lines, preferably humanpluripotent cells and cell lines, which are derived from DNA of entirelymale or female original, have numerous therapeutic and diagnosticapplications. Most especially, such pluripotent cells may be used forcell transplantation therapies or gene therapy (if geneticallymodified). Human ES cells have application in the treatment of numerousdisease conditions.

In this regard, it is known that mouse embryonic stem (ES) cells arecapable of differentiating into almost any cell type, e.g.,hematopoietic stem cells. Therefore, human or other mammalianpluripotent (ES) cells produced according to the invention shouldpossess similar differentiation capacity. The pluripotent cellsaccording to the invention will be induced to differentiate to obtainthe desired cell types according to known methods. For example, human EScells produced according to the invention may be induced todifferentiate into hematopoietic stem cells, muscle cells, cardiacmuscle cells, liver cells, cartilage cells, epithelial cells, urinarytract cells, etc., by culturing such cells in differentiation medium andunder conditions which provide for cell differentiation. Medium andmethods which result in the differentiation of ES cells are known in theart as are suitable culturing conditions.

For example, Palacios et al., Proc. Natl. Acad. Sci., USA, 92:7530-7537(1995) teach the production of hematopoietic stem cells from anembryonic cell line by subjecting stem cells to an induction procedurecomprising initially culturing aggregates of such cells in a suspensionculture medium lacking retinoic acid followed by culturing in the samemedium containing retinoic acid, followed by transferral of cellaggregates to a substrate which provides for cell attachment.

Moreover, Pedersen, J. Reprod. Fertil. Dev., 6:543-552 (1994) is areview article which references numerous articles disclosing methods forin vitro differentiation of embryonic stem cells to produce variousdifferentiated cell types including hematopoietic cells, muscle, cardiacmuscle, nerve cells, among others.

Further, Bain et al., Dev. Biol., 168:342-357 (1995) teach in vitrodifferentiation of embryonic stem cells to produce neural cells whichpossess neuronal properties. These references are exemplary of reportedmethods for obtaining differentiated cells from embryonic or stem cells.These references and in particular the disclosures therein relating tomethods for differentiating embryonic stem cells are incorporated byreference in their entirety herein.

Thus, using known methods and culture medium, one skilled in the art mayculture the subject ES cells, including genetically engineered ortransgenic ES cells, to obtain desired differentiated cell types, e.g.,neural cells, muscle cells, hematopoietic cells, etc.

Pluripotent cells produced by the invention may be used to obtain anydesired differentiated cell type. Therapeutic usages of differentiatedhuman cells are unparalleled. For example, human hematopoietic stemcells may be used in medical treatments requiring bone marrowtransplantation. Such procedures are used to treat many diseases, e.g.,late stage cancers such as ovarian cancer and leukemia, as well asdiseases that compromise the immune system, such as AIDS. Hematopoieticstem cells can be obtained, e.g., by incorporating male or female DNAderived from a male or female cancer or AIDS patient with an enucleatedoocyte, obtaining pluripotent cells as described above, and culturingsuch cells under conditions which favor differentiation, untilhematopoietic stem cells are obtained. Such hematopoietic cells may beused in the treatment of diseases including cancer and AIDS.

Alternatively, the subject pluripotent cells may be used to treat apatient with a neurological disorder by culturing such cells underdifferentiation conditions that produce neural cell lines. Specificdiseases treatable by transplantation of such human neural cellsinclude, by way of example, Parkinson's disease, Alzheimer's disease,ALS and cerebral palsy, among others. In the specific case ofParkinson's disease, it has been demonstrated that transplanted fetalbrain neural cells make the proper connections with surrounding cellsand produce dopamine. This can result in long-term reversal ofParkinson's disease symptoms.

The great advantage of the subject invention is that it provides anessentially limitless supply of pluripotent, preferably pluripotenthuman cells that can be used to produce differentiated cells suitablefor transplantation. Such cells should alleviate the significant problemassociated with current transplantation methods, i.e., rejection of thetransplanted tissue which may occur because of host-vs-graft orgraft-vs-host rejection. Conventionally, rejection is prevented orreduced by the administration of anti-rejection drugs such ascyclosporine. However, such drugs have significant adverse side-effects,e.g., immunosuppression, carcinogenic properties, as well as being veryexpensive. The present invention should eliminate, or at least greatlyreduce, the need for anti-rejection drugs.

Other diseases and conditions treatable by cell therapy include, by wayof example, spinal cord injuries, multiple sclerosis, musculardystrophy, diabetes, liver diseases, i.e., hypercholesterolemia, heartdiseases, cartilage replacement, burns, foot ulcers, gastrointestinaldiseases, vascular diseases, kidney disease, urinary tract disease, andaging related diseases and conditions.

This methodology can be used to replace defective genes, e.g., defectiveimmune system genes, cystic fibrosis genes, or to introduce genes whichresult in the expression of therapeutically beneficial proteins such asgrowth factors, lymphokines, cytokines, enzymes, etc. For example, thegene encoding brain derived growth factor may be introduced into humanpluripotent cells produced according to the invention, the cellsdifferentiated into neural cells and the cells transplanted into aParkinson's patient to retard the loss of neural cells during suchdisease.

Previously, cell types transfected with BDNF varied from primary cellsto immortalized cell lines, either neural or non-neural (myoblast andfibroblast) derived cells. For example, astrocytes have been transfectedwith BDNF gene using retroviral vectors, and the cells grafted into arat model of Parkinson's disease (Yoshimoto et al., Brain Research,691:25-36 (1995)).

This ex vivo therapy reduced Parkinson's-like symptoms in the rats up to45% 32 days after transfer. Also, the tyrosine hydroxylase gene has beenplaced into astrocytes with similar results (Lundberg et al., Develop.Neurol., 139:39-53 (1996) and references cited therein).

However, such ex vivo systems have problems. In particular, retroviralvectors currently used are down-regulated in vivo and the transgene isonly transiently expressed (review by Mulligan, Science, 260:926-932(1993)). Also, such studies used primary cells, astrocytes, which havefinite life span and replicate slowly. Such properties adversely affectthe rate of transfection and impede selection of stably transfectedcells. Moreover, it is almost impossible to propagate a large populationof gene targeted primary cells to be used in homologous recombinationtechniques. By contrast, the difficulties associated with retroviralsystems should be eliminated by the use of the methods herein.

Genes which may be introduced into the subject pluripotent cellsinclude, by way of example, epidermal growth factor, basic fibroblastgrowth factor, glial derived neurotrophic growth factor, insulin-likegrowth factor (I and II), neurotrophin-3, neurotrophin-4/5, ciliaryneurotrophic factor, AFT-1, cytokine genes (interleukins, interferons,colony stimulating factors, tumor necrosis factors (alpha and beta),etc.), genes encoding therapeutic enzymes, etc.

In addition to the use of human pluripotent cells and cells derivedtherefrom in cell, tissue and organ transplantation, the presentinvention also includes the use of non-human cells in the treatment ofhuman diseases. For example, non-human primate pluripotent cellsproduced according to the invention should be useful for treatment ofhuman disease conditions where cell, tissue or organ transplantation iswarranted (given the phylogenetic closeness of primates and humans(immunogenicity should be less of a concern.) In general, pluripotentcells and differentiated cells derived therefrom produced according tothe present invention can be used within the same species (autologous,syngenic or allografts) or across species (xenografts). For example,brain cells derived from bovine or porcine pluripotent cells may be usedto treat Parkinson's disease.

Also, the subject pluripotent ES cells, preferably human cells, may beused as an in vitro model of differentiation, in particular for thestudy of genes which are involved in the regulation of earlydevelopment. Also, differentiated cell tissues and organs produced usingthe subject ES cells may be used in drug studies.

Further, the subject ES cells or differentiated cells derived therefrommay be used as nuclear donors for the production of other ES cells andcell colonies, or in the case of non-human cells, for the production ofcloned animals.

Still further, pluripotent cells obtained according to the invention maybe used to identify proteins and genes that are involved inembryogenesis. This can be effected e.g., by differential expression,i.e., by comparing mRNA's that are expressed in pluripotent cellsprovided according to the invention to mRNAs that are expressed as thesecells differentiate in to different cell types, e.g., neural cells,myocardiocytes, other muscle cells, skin cells, etc. Thereby, it may bepossible to determine what genes are involved in differentiation ofspecific cell types.

Also, it is another object of the invention to expose pluripotent celllines produced according to the invention to cocktails of differentgrowth factors, at different concentrations so as to identify conditionsthat induce the production and proliferation of desired differentiatedcell types.

In order to more clearly describe the subject invention, the followingexample is provided.

Example 1 Production of Pluripotent Cells by Activation of BovineOocytes Containing DNA of Female Origin

The example describes one method of producing pluripotent cells byandrogenetic or gynogenetic activation and production of embryos. Inparticular, this example describes the production of pluripotent cellsfrom parthenogenetically activated bovine oocytes (gynogeneticactivation of bovine oocytes containing all female DNA effected usingionomycin and DMAP). As described below, and substantiated by FIGS. 1through 10, this procedure resulted in the production of an embryohaving a discernible trophectoderm and inner cell mass, the inner cellmass of which, when cultured on a mouse fetal fibroblast feeder layergave rise to pluripotent cells which produce differentiated cells (Seeespecially FIG. 10).

Materials and Methods Used in Example

Media and Solutions Hyaluronidase (0.01% solution) Hyaluronidase (SigmaH-3506) 1 mg DPBS 1 ml TL Hepes NaCNaCl (Sigma S-5886) 666 mg KCl (SigmaP-5404) 24 mg NaHCO₃ (Sigma S-5761) 16.8 mg NaH₂PO₄—H₂O (Sigma S-9638)4.76 mg Hepes (Sigma H-9136) 240 mg Na Lactate (60% syrup) (SigmaL-1375) 186 μl Phenol Red (0.5% solution) (Sigma P-0290) 100 μlCaCl₂-₂H₂O (Sigma C-7902) 300 mg MgCl₂6H₂O (Sigma M-2393) 5 mg Embryotransfer H₂O (Sigma W-1503) enough to make final volume 100 ml Adjust pHto 7.3-7.4 Filter with 0.2 μm filter and place in 50 ml capped bottle,label and date. Store buffer at 4° C. HECM Hepes NaCl (Sigma S-5886)0.662 g KCl (Sigma P-5405) 0.0239 g CaCl₂—2H₂O (Sigma C-7902) 0.0294 gMgCl₂—6H₂O (Sigma M-2393) 0.0102 g BME-amino acids (Sigma B-6766) 1 mlNa Lactate (Sigma L-1375) 1.4 ml Na Pyruvate (Sigma P-2256) 0.011 gNaHCO₃ (Sigma S-5761) 0.168 g Hepes (Sigma H-9136) 0.238 g Phenol Red(Sigma P-0290) 0.1 ml Pen/Strep (Sigma P-3539) 1 ml BSA fraction V(Sigma A-4503) 0.3 g Embryo transfer H₂O (Sigma W-1503) enough to makefinal volume 100 ml Osmolarity must be 275 +/− 10 mOsm/kg Adjust pH to7.3-7.4 Filter with 0.2 μm filter and place in 50 ml capped bottle,label and date. Store buffer at 4° C. ACM Media NaCl (Sigma S-5886)0.290 g KCl (Sigma P-5405) 0.011 g CaCl₂—2H₂O (Sigma C-7902) 0.002 gNaHCO₃ (Sigma S-5761) 0.105 g Na Lactate (Sigma L-4263) 7 μl Pyruvatestock 1 ml BME amino acids (Sigma B-6766) 1 ml MEM amino acids (SigmaM-7145) 0.5 ml L-Glutamine (Sigma G-5763) 7.3 mg Pen/Strep (SigmaP-3539) 0.5 μl BSA fraction V (Sigma A-4503) 150 g Embryo transfer H₂O(Sigma W-1503) enough to make final volume 50 ml Adjust pH to 7.3-7.4Filter with 0.2 μm filter and place in 50 ml capped bottle, label anddate. Store buffer at 4° C. Pyruvate Stock Pyruvic Acid (Sigma P-2256)2.2 mg Embryo transfer H₂O (Sigma W-1503) 1 ml Label and store at −20°C. in 1 ml aliquots. Ionomycin Ionomycin (Calbiochem 407952 or 1 mg(Sigma 1-0634) Dimethyl Sulfoxide (Sigma D-8799) 267.6 μl Label andstore at −4° C. in 5 μl aliquots. Z1 TL Hepes + 1 mg/ml BSA (fractionV), pH to 7.2-7.4. Filter, label and store at 4° C. in a cappedcontainer. DMAP 200 mM (100X stock) DMAP (Sigma D-2629) 1 g PBS (Gibco21600-051) 30 ml Heat in boiling water bath to dissolve DMAP. Aliquot 20μl into small eppendorf tubes, label and date. Store aliquots at −20° C.Mouse Fetal Fibroblasts Feeder Layers Mouse fetal fibroblasts are gammairradiated for five minutes and plated at 1 × 10⁶ cells per ml ofALPHA-MEM culture media. Leave overnight or until cells are plated andchange media to desired culture media. ALPHA-MEM Culture Media ALPHA-MEM(Gibco 12-169F) 500 ml Fetal Calf Serum (HyClone A-111-D) 50 mlAntibiotic/Antimicotic (Sigma A-7292) 2 ml 2-Mercaptoethano 1 (Gibco21985-023) 1.4 ml L-Glutamine Stock 5 ml Tylosine Tartrate (SigmaG-5763) 0.5 ml L-Glutamine Stock L-Glutamine (Sigma G-5763) 1-5 g DPBS50 ml Filter and aliquot 5.5-6.0 ml in 15 ml tubes. Label and freeze onside.

Procedures—Oocyte Preparation

Bovine oocytes were stripped eighteen hours post maturation using 0.01%solution of hyaluronidase (1 mg/ml) contained in TL Hepes media.Afterward, the stripped oocytes are rinsed using HECM Hepes or TL Hepes.The resultant stripped, rinsed oocytes are then stored or used directlyfor activation. Storage can be effected, e.g., in ACM at 37° C. and fivepercent CO₂ until activation. Preferably, the ACM is equilibrated at 37°C. and five percent CO₂ for about two to three hours before usage foroocyte storage.

Oocyte Activation

An appropriate activation protocol is used that results in theproduction of embryos having a discernible trophectoderm and inner cellmass. As previously disclosed, various methods can be used.

In particular, the inventors elected to effect activation by placingoocytes (prepared as above) in Z1 media containing 5 μM of ionomycin forfour minutes. This media was prepared by diluting 2 μL of 5 mM ionomycinin 2 ml of Z1 medium.

Afterward, the oocytes were washed using HECM Hepes, and are thenincubated in DMAP/ACM medium for three to four hours. This medium wasprepared by dilution of 5 μL of 200 mM DMAP in 500 μl of ACM, withACM/DMAP preferably being equilibrated at 37° C. and five percent CO₂ inair for two to three hours prior to usage.

After incubation in DMAP/ACM, oocytes were washed four times in HECMHepes. The washed oocytes were then placed in ACM on a mouse fibroblastfeeder layer prepared as described above. The ACM media was againequilibrated at 37° C. and five percent CO₂ in air for two to threehours prior to use.

This resulted in the production of blastocysts having a discernibletrophectoderm and inner cell mass (See FIGS. 1 through 10). Seven tonine days post activation, the blastocysts were dissected using 30gauge/1 inch needles and placed on mouse fetal fibroblast feeder layersin ALPHA-MEM tissue culture medium. The cells were incubated thereon at37° C. and five percent CO₂ for one week.

The medium was changed every two to three days following said week ofincubation.

The cells were passaged onto new feeder layers manually. This waseffected by culturing the colonies and pipetting directly the piecesonto new feeder layers. This procedure can be repeated by change ofmedium and repeated passaging indefinitely to produce pluripotent cellsthat give rise to differentiated cells. The efficacy of the describedmethods is substantiated by FIGS. 1 through 10. In particular, FIG. 10shows a pluripotent cell colony produced from a blastocyst produced bygynogenetic (parthenogenic) activation as described above, resulted in apluripotent (stem cell) colony with differentiated cells being observedat the edge of the colony.

Example 2 Production of Parthogenic Primate Primordial Stem Cells(PPSCS) Materials and Methods

1-Cynomolgous Monkey (Macaca fascicularis) were superovulated using asingle injection of 1000 IU of pregnant mare's serum gonadtrophin (PMSG)and 500 IU of human chorionic gonadoprophin (hCG) four days later.

2-Ovaries were retrieved by laparotomy and oocytes dissected from thefollicles and placed in maturation media 36 to 48 hrs after (hCG).Maturation media consisted of medium-199 (Gibco BRL) with Earle'sbalanced salt solution supplemented with 20% fetal bovine serum, 10IU/ml of PMSG, 10 IU/ml of hCG, 0.05 mg/ml of penicillin G and 0.075mg/ml of streptomycin sulfate (Hong, 1999).

3-Oocyte Activation. After 40 hrs in maturation, metaphase II eggs wereplaced in 10 micromoles of Ionomycin followed incubation in 200 mM6-DMAP (dimethylaminopurine) for 3 to 4 hrs.

4-Embryo Culture. Commercially available embryo culture media ‘Cooks’was used (modified SOF). Embryos were cultured with a co-culture ofmitotically inactivated mouse embryonic fibroblasts as feeder layer.

5-Isolation of Inner Cell Mass

-   -   1—Upon development to blastocyst, embryos were placed in a        buffered solution of 3% pronase for 2 minutes to digest zona        pellucida.    -   2—Blastocysts were then rinsed in buffered solution and moved to        solution of G1 culture media and polyclonal antibodies        (antihuman whole serum) 1:3 dilution for 30 minutes.    -   3—Embryos were rinsed 5 times in a buffered solution.    -   4—Embryos were moved into a solution of G1 culture media and        guinea pig complement 1:3 dilution for 30 minutes.    -   5—Remaining of the embryos (dead trophoblast cells and ICM) were        rinsed 5 times in buffered solution the Inner Cell Mass (ICM)        was isolated and placed on top of a mouse embryonic fibroblast        feeder layer for isolation and growth of Primordial Stem Cells        (PSC's).

Results

We have obtained 450 eggs total, after maturation, 224 were still atgerminal vesicle stage (GV=no maturation), 79 were dead, 56 were atmetaphase one (MI) and 91 at metaphase two (MII).

We have parthenogenetically activated all of them. As expected, therewas no cleavage on the GV group, 32% cleavage on the MI and 57% on theMII. When put in culture, 7 embryos developed to the blastocyst stage(See FIG. 11).

After attempting to establish ES-like culture cells, four Inner cellmasses attached nicely one differentiated immediately, and out of thethree remaining, one cell line was obtained. This cell line is calledCyno 1 (FIG. 12). This cell line before and after immunosurgery is shownin FIGS. 12 and 13. FIG. 14 shows the Cyno 1 cell line five days afterplating.

FIG. 15 shows the Cyno 1 cell line growing on top of a mouse fibroblastfeeder layer. These cells show typical morphology ofpluripotent-embryonic-cells such as small nuclear cytoplasmic ratio andthe presence of cytoplasmic granules.

These cells were maintained in an undifferentiated state for a period ofup to 11 months. This is evidenced by screening of such cells afterprolonged culturing for the expression of a cell marker characteristicof undifferentiated cells, Alkaline Phosphatase. As expected, cells werepositive on passage 3 and on passage 5.

Cyno-1 cells displayed many features that are typical for ES cells:Morphologically, the cells had a small cytoplasmic/nuclear ratio,numerous and prominent nucleoli and cytoplasmic lipid bodies. They couldbe extensively propagated in vitro while maintaining theirundifferentiated state. These cells tested positive for alkalinephosphatase, and were immunoreactively positive for SSEA-4, TRA 1-60,TRA 1-81 and Oct-4, and negative for SSEA-1 and SSEA-3. The fact thatCyno-1 cells stain negatively for SSEA-3 physically distinguishes themfrom other primate and human stem cells described previously. Thomson etal., Biol. Reprod. 55(2):254-9 (1996); Thomson et al., Science282(5391):1145-7 (1998). In addition, karyotyping revealed 40+2chromosomes as is expected for Macaca fascicularis (data not shown).

The fact that these cells maintain their pluripotency is also shown bytheir spontaneous differentiation into many differentiated cell typesafter being placed in tissue culture in the absence of a feeder layer.Differentiation of Cyno-1 cells was induced by allowing the cells toovergrow and by modifying culture conditions, i.e., by isolating thecells from the mouse feeder layer and culturing them in the presence ofDMEM with 15% fetal calf serum, in some instances 1000 IU of LIF wasadded to the media. In the days following, the cells were observed todifferentiate into cuboidal and ciliated epithelium, fibroblasts,beating myocardial cells, smooth muscle cells and cytokeratine-positivecells as well as neuronal cells. Two colonies of beating myocardialcells were observed in one well of a 4-well tissue culture plate.

Cyno-1-derived neural cells proliferated readily and expressed the CNSstem cell marker nestin (data not shown). They were also observed todifferentiate into astrocytes or neurons depending on the cultureconditions. The most remarkable differentiation observed in Cynol cellswas obtained when derived neural precursors were exposed to definedmorphogenic factors. In this case, up to 25% of midbrain dopamineneurons was observed. This is a specialized population of neurons, whoseefficient generation from primate embryonic stem cells has not beenreported previously. Neuronal identity and function was confirmed byHPLC analysis measuring in vitro release of the neurotransmittersdopamine and serotonin (data not shown). Cyno-1 derived neuronsexhibited both basal and KCl-evoked synaptic release of dopamine andserotonin.

In vivo random differentiation of Cyno-1 cells was tested by injectingthem into the peritoneal cavity of immunocompromised SCID mice.Teratomas were isolated 8 and 15 weeks after injection and subjected tohistological analysis. Derivatives of all three germ layers wereobserved including cartilage, muscle and bone (mesoderm), neurons, skinand hair follicles (ectoderm) and intestinal epithelia (endoderm). Thepresence of mature tissues and low frequency of mitotic figures in thesetumors indicated their benign nature.

Telomerase activity is often correlated with replicative immortality.Telomerase is typically expressed in germ cells, cancer cells and avariety of stem cells, including embryonic stem cells, but absent inmost somatic cell type. Undifferentiated Cyno-1 cells displayed highlevels of telomerase activity as detected by the TRAP assay (TRAPEZEKit, Intergen, N.Y.). However, no telomerase activity could be detectedin differentiated progeny of Cyno-1 cells (data not shown). These dataindicate a physiologically normal control of telomerase activity inCyno-1 cells.

To determine whether differentiated cells of various somatic celllineages were observed from the differentiating PPSCs, we extracted mRNAfrom differentiated cell cultures, performed RT-PCR, using humansequence primers specific for various differentiated cell types. Asshown in FIG. 16, transcripts of a predicted size for themesodermally-derived transcripts brachyury and skeletal muscle myosinheavy polypeptide 2 were observed. The transcript sonic hedgehogessential for endoderm development was observed. In addition, theneuron-specific ectoderm marker enolase was observed as well as keratin(not shown) as markers of ectodermally derived cells. These PCR productswere not observed in the mouse feeder layer controls or in the absenceof reverse transcriptase.

To establish that the imprinting status of parthogenetic PPSCs isdifferent than that of di-parental PPSCs we looked at the expression ofseveral imprinted genes. Genes that are mono-allelically expressed fromthe paternal allele, would not be expected to be expressed inparthogenetic cells, as these cells are derived exclusively from thematernal genome. The Snrpn gene is mono-allelically expressed from thepaternal allele in mouse blastocyst inner cell mass [Szabo, P E andMann, J R; Genes & Development 9:3097-3108 (1995)]. We looked at theexpression of this gene in the parthogenetic Macaca facicularis PPSCsand found that the expression was undetectable by RT-PCR, whereas underidentical conditions, this gene is readily detected in fibroblast cellcultures from the same species. The Snrpn gene is expected to beexpressed in diparental PPSCs, as these cells contain a paternal allele.In FIG. 17, the expected size RT-PCR product for the Snrpn gene is 260bp. Thus, Cyno-1 cells may provide a novel tool for assessing theeffects of genomic imprinting on cell differentiation and functionduring development in primates.

DNA profiling of Cyno-1 cells was performed to confirm the identity ofthese cells with respect to the donor animal. Total genomic DNA from acynomolgus monkey cell donor #5571 (Buttercup) and from a preparation ofcultured stem cells (Cyno-1; derived from Buttercup) were genotyped andcompared using 7 simple sequence repeat (SSR) human markers (ResearchGenetics, Inc.; Huntsville, Ala.) that had been shown previously toamplify monkey DNA and to discriminate between two individuals. Themarkers represent 7 different chromosomes (#3,6,7,10,11,16 & 17) and inall cases except one (marker D16S403), alleles for Buttercup wereidentical in number and size to the alleles for the Cyno-1 cells. Anadditional test was performed on DNA from Buttercup and from the Cyno-1cells (as well as 2 control animals). Micro SSPTM Generic HLA Class IIDNA typing was performed in a 96 well tray format through the WakeForest University—Baptist Medical Center Histocompatibility Laboratory.The data demonstrated that embryonic stem cells Cyno-1 and somatic cellsfrom Buttercup were indistinguishable from each other and thereforeshould be considered autologous (data not shown).

The histocompatibility antigen profile of Cyno-1-derived neurons wasinvestigated and compared to lymphocytes from the oocyte donor byinvestigating polymorphic genes within the major histocompatibilitycomplex (MHC) that encode class I and class II cell surface proteins.These proteins present immunogenic peptides to CD8⁺ and CD4⁺ T cells,respectively. We have analyzed the Cyno-1-derived neural cells by flowcytometry for the expression of Mafa (MHC of M. fascicularis) class Iand class II antigens. Peripheral blood lymphocytes (PBLs) from theoriginal cell donor expressed class I and class II antigens detected byantibodies specific for monomorphic HLA-A,B,C and HLA-DR antigens,respectively (data not shown). Seventy-five percent of PBLs werepositive for class I and 14% of PBLs were positive for class II.However, Cyno-1-derived neural cells were negative for both Mafa class Iand class II antigens, consistent with observations that these CNS celltypes are class I- and class II-negative in normal murine centralnervous system. Altintas, A., et al., Differential Expression of H-2Kand H-2D in the Central Nervous System of Mice Infected with Theiler'svirus, J. Immunol., 151(5):2803-12 (1993); Rodriguez, M., M. L. Pierce,and E. A. Howie, Immune Response Gene Products (Ia Antigens) on Glialand Endothelial Cells in Virus-Induced Demyelination, J. Immunol.,138(10):3438-42 (1987).

Viral infection or treatment with interferon-gamma (IFNγ) stimulatesupregulation of class I and class II expression by murine CNS cells. Theability of IFNγ to upregulate class I and class II expression byCyno-1-derived neural cells was investigated by pre-culturing thesecells with IFNγ (40 ng/ml) overnight prior to staining and flowcytometry. Pre-treatment of Cyno-1 derived cells resulted in classI-specific staining with an intensity that was comparable to staining ofnormal human PBLs. However, IFNγ treatment did not increase class IIexpression. These results support the prediction that an in vivoinflammatory response, expectedly involving IFNγ expression, wouldupregulate class I expression on transplanted Cyno-1-derived neuralcells. Accordingly, in the event of transplantation into a non-isogenicanimal, these cells should not escape surveillance by CD8⁺ cytotoxic Tlymphocytes.

In conclusion, we have generated a primate parthenogenetic cell linewith ES-like properties that can be propagated in vitro in anundifferentiated state for at least 11 months. The in vitro derivationof large numbers of specific cell lineages from Cyno-1 cells, includingthe generation of unlimited numbers of dopaminergic neurons is ofparticular interest. Clinical transplantation of specific fetal neuronshas shown promise in the treatment of Parkinson's and Huntington'sdisease but obtaining such cells from animals or human fetal brainremains problematic. Neurons derived in vitro from a renewable sourcesuch as CNS precursors, embryonic, or parthenogenetic animal or humanstem cells, could alleviate some of the ethical and technical concernsof human cell therapy. In addition, Cyno-1 cells may be useful in the invitro study of imprinting, early development, and for the isolation ofembryonic proteins and cell components, useful in the reprogramming ofhuman cells.

The protocols described here for parthenogenetic activation of humaneggs, coupled with the derivation of primate parthenogenetic stem cells,are likely to be applicable for the generation of human parthenogeneticstem cells. As our understanding of fate determination advances, suchstem cells may constitute a source of specialized cell types for a widerange of therapeutic applications.

While the invention has been described with respect to certain specificembodiments, it will be appreciated that many modifications and changesthereof may be made by those skilled in the art without departing fromthe spirit of the invention. It is intended, therefore, by the appendedclaims to cover all modifications and changes that fall within the truespirit and scope of the invention.

What is claimed is:
 1. A method for producing pluripotent (ES) cells that can be used to produce differentiated cells and tissues comprising: (a) obtaining a haploid cell in metaphase II that comprises DNA derived from a single individual male or female, which optionally may be genetically modified; (b) activating said haploid cell by a method selected from the group consisting of: (1) conditions that do not result in second polar body extrusion; (2) conditions that provide for polar body extrusion but in the presence of an agent that inhibits polar body extrusion; and (3) conditions that prevent the initial cleavage, and culturing said activated cell to produce a gynogenetic or androgenetic embryo comprising a discernible trophectoderm and an inner cell mass; (c) isolating said inner cell mass or cells therefrom and transferring said inner cell mass or cells to an in vitro media that inhibits differentiation of said inner cell mass derived therefrom; and (d) culturing said inner cell mass cells or cells derived therefrom to maintain said cells in an undifferentiated pluripotent state.
 2. The method of claim 1, wherein the metaphase II cell is an oocyte or blastomere.
 3. The method of claim 2, wherein the haploid cell is a human, non-human primate, bovine, porcine, or ovine oocyte or blastomere.
 4. The method of claim 3, wherein the haploid DNA derived from a single individual is human, bovine, primate, ovine, or porcine.
 5. The method of claim 4, wherein the cell is a human or bovine oocyte and the haploid DNA is human DNA.
 6. The method of claim 1, where said activation conditions include the use of DMAP (phosphorylation inhibitor) or other compound that inhibits second polar body extrusion.
 7. The method of claim 1, wherein activation conditions include use of a compound that inhibits microfilament or protein production.
 8. The method of claim 7, wherein said compound is cycloheximide or cytochalasin B.
 9. The method of claim 1, wherein the haploid DNA is of a female origin.
 10. The method of claim 1, wherein haploid DNA is of male origin.
 11. The method of claim 1, wherein the haploid cells are human oocytes containing human male or female DNA.
 12. The method of claim 1, wherein said cultured cells of (d) are allowed to differentiate.
 13. The method of claim 1, wherein said cells are implanted at a desired site in vivo that is to be engrafted with cells or tissue.
 14. The method of claim 13 wherein said cells are implanted in an immunocompromised non-human animal.
 15. The method of claim 13, wherein said site is a wound, a joint, muscle, bone, or the central nervous system.
 16. The method of claim 1, wherein the cell obtained by (d) is genetically modified. 